Global Eukaryotic Microcarrier™ (GEM) FAQs
Why do we call the GEM technology 3D?
How are the cells dissociated from the GEM?
Is there any influence of the paramagnetic particles on the physiology of the cells?
Do the cells grow inside the GEM?
What GEM protein coatings are available?
How are the cells counted while in culture?
How do you passage the cells?
How do you know if the GEMs are confluent and how do you measure the confluence?
Can the cells be frozen down?
What is the proper method for visualizing the GEMs with cells using microscope?
Can the yield be increased by adding additional GEMs during the inoculation phase?
How do I know which coating is appropriate for my cell type?
Can the cells be directly assayed on the GEM?
Why do we call the GEM technology 3D?
"3D" is a reference to the sphere on which cells grow. With "3D" we mean that cell culture on the GEM is more physiological. Cells on GEM are healthier because they are passaged and trypsinized about 3 times less often as compared to growth in a T-flask. It has also been demonstrated that some primary cells have an improved in vivo phenotype when grown on the GEM substrate (see Justice et al. 2009 in Drug Discov Today).
How are the cells dissociated from the GEM?
Cells may be dissociated from the GEM with traditional cell dissociation agents such as trypsin, Tryple, Accutase™ (from Global Cell Solutions), etc. The remaining particles can be drawn to the bottom of the cell culture tube with a CubeMagnet™. The cells can then be harvested and used in downstream applications.
Is there any influence of the paramagnetic particles on the physiology of the cells?
No influence of the paramagnetic particles has been measured so far on cell growth or viability. The paramagnetic particles are actually silicated iron particles that do not possess a magnetic field. They are embedded in the alginate core and do not contact the cells directly.
Do the cells grow inside the GEM?
No, the cells grow on the covalently bound protein layer on the outside of the GEM. There are many different protein coatings and other adhesion molecules that are available. Due to this property the GEM supports many adherent cell types.
What GEM protein coatings are available?
- Gelatin
- Laminin
- Basement Membrane
- Collagen I
- Collagen IV
- Fibronectin
- Poly-d-lysine
- Custom coatings are available at Global Cell Solutions on a contracted basis.
How are the cells counted while in culture?
Cells are easily counted in one of two ways. The first way is to dissociate the cells from the GEM with agents such as trypsin, Tryple, Accutase™ (from Global Cell Solutions), etc. Once the cells are dissociated they may be counted using any traditional method (e.g. hemocytometer, flow cytometer, trypan blue, etc.). The second way is to lyse the cells while attached to the GEM, which releases the nuclei. The nuclei may be treated with propidium iodide and counted using the NucleoCounter from ChemoTec.
How do you passage the cells?
There are two ways we can passage the cells using the GEM technology. The first way is to trypsinize an aliquot of GEM using Trypsin, Tryple, Accutase™ (from Global Cell Solutions), etc.; these agents release the cells into suspension. The magnetic particles may be pulled down using a CubeMagnet™; the cell suspension may be pipetted off, and added to fresh GEMs. The second way we can amplify the culture is to add GEM confluent with cells to fresh GEM for inoculation in a new LeviTube..
How do you know if the GEMs are confluent and how do you measure the confluence?
To analyze the confluence the cells may be stained while attached to the GEM with Hoechst. Simply count the number of nuclei present on the surface of the GEM to get a relative idea of the cell density. You may also visualize the GEM-cell complex under a light microscope. The GEM will appear as a large round sphere with grayish-brown particulates inside. The cells will be transparent circles on the outside of the GEM sphere.
Can the cells be frozen down?
The cells can be harvested and frozen down using traditional cryopreservation protocols or the cells can be frozen while attached to the GEM. We recommend that you freeze your cells while they are attached to the GEM, this limits cellular exposure to harsh chemicals such as trypsin. The cryopreservation protocol is outlined in the GEM Manual provided by Global Cell Solutions.
What is the proper method for visualizing the GEMs with cells using microscope?
Cells can be imaged both live and fixed on the GEM. The GEM-cell complex may be viewed using a light microscope. The GEM will appear as a large round sphere with grayish-brown particulates inside. These particles are the silicated iron particles. The cells will be transparent circles on the outside of the GEM sphere. At 20X-40X you should be able to see cells attached to GEM. You may stain the cells with Hoechst or a membrane marker to see individual cells. You may use standard staining techniques to visualize the cells. The CubeMagnet may be used to help manipulate the GEMs into the proper position.
Can the yield be increased by adding additional GEMs during the inoculation phase?
Yes, more GEMs may be used during the inoculation phase. The more surface area available for growth will result in a higher final yield. The limiting factor will be the medium. The more GEM and cells used in the inoculation phase will lead to more cells proliferating on the surface of the GEM and faster consumption of the medium components essential for growth. The optimum cell to GEM ratio should be identified for each cell type cultured.
How do I know which coating is appropriate for my cell type?
If you are unsure about the proper coating to use you may try an Adhesion Assay kit. This kit contains six different GEM coatings. The kit is used to determine cell affinity for the different GEM coating and will not indicate final cell yields. You may also contact Hamilton or Global Cell Solutions to see if we have data for the cell type you are interested in culturing.
Can the cells be directly assayed on the GEM?
Most assays may be performed with the cells attached to the GEM. The user may use protocols that are typically used for traditional methods on the GEM. Since the cells have not been exposed to harsh chemicals such as trypsin, they may perform better in assays since they are less disturbed by chemicals.
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